a300 110a  (Bethyl)

Journal: International Journal of Molecular Sciences

Article Title: ESCO2 Interacts with TRF1/2 and Facilitates Telomere Maintenance

doi: 10.3390/ijms27062635

Figure Lengend Snippet: ESCO2 deficiency results in telomere abnormalities. ( A ) HeLa1.2.11 cells transfected with indicated siRNAs were analyzed by Q-FISH using a FITC-labeled telomere PNA probe. DAPI was used to stain the nuclei. The histograms show the distribution of relative telomere length presented as fluorescence intensity (TFU, telomere fluorescence unit); the red lines mark the mean telomere signal intensity. n indicates the total number of telomere signals detected. Error bars indicate standard errors. *** p < 0.001. ( B ) Examples of telomere abnormalities from ( A ) observed in a telomere FISH assay (upper panel). The incidence of telomere abnormalities in cells lacking ESCO1 or ESCO2 is shown in the bottom panel. ( C ) Sankey diagram showing KEGG pathway analysis of high-confidence proteins associated with ESCO2. Pathway enrichment was performed based on high-confidence ESCO2-interacting proteins, and the results are visualized as a Sankey diagram to illustrate the functional distribution across different KEGG pathways. ( D ) Chord diagram showing KEGG pathway analysis of DNA replication- and repair-related pathways and their associated proteins. Diagram visualizes the relationship between key proteins and their corresponding pathways involved in DNA replication and repair. ( E ) Selected lists of ESCO2-associated proteins analyzed by mass spectrometry. ( F ) Western blot performed to determine ESCO2 and BLM siRNA-knockdown efficiency in HeLa1.2.11 cells. ( G ) Representative images of metaphase telomere FISH in cells from ( G ) (upper panel). The incidence of telomere abnormalities is shown in the bottom panel.

Article Snippet: The antibodies used in this study were: 53BP1 (NB100-904; Novus Biologicals, Littleton, CO, USA), Flag (F3165; Sigma, St. Louis, MO, USA), BLM (A300-110A, Bethyl, Boston, MA, USA), ESCO2 (ab86003; Abcam, Cambridge, UK), Myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and vinculin (V9131; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Transfection, Labeling, Staining, Fluorescence, Functional Assay, Mass Spectrometry, Western Blot, Knockdown

Journal: Communications Biology

Article Title: BLM and FANCJ role in the response to G-quadruplex-dependent telomeric replicative stress

doi: 10.1038/s42003-025-09367-z

Figure Lengend Snippet: a The diagram shows the workflow on a timeline: on the day after cell seeding, cells were treated with RHPS4 (0.2 and/or 0.5 μM). At 48 h after treatment, cells were again treated with RHPS4 (0.2 and/or 0.5 μM) or harvested and counted. At 72 h after the first treatment, the cell viability was checked, and finally at 96 h after the first treatment, the cells were harvested and processed. b Representative images showing U251MG cells stained by immunofluorescence using anti-BLM (red signals), and anti-TRF1 or anti-TRF2 (green signals) antibodies. Merged images allow visualization of colocalizing dots (yellow signals). Yellow arrows indicate BLM and TRF1-TRF2 colocalizations. c Quantification of the colocalizations between BLM and TRF1 or TRF2 proteins in U251MG cell line upon RHPS4 treatment (0.2 and 0.5 μM). d , e Sensitivity of U251MG and BLM −/− cell lines to RHPS4 concentrations ranging from 0.01 to 2 μM (0.01; 0.125; 0.25; 0.5; 1; 2 μM), evaluated 96 h after the first treatment. Mitomycin C (MMC) was used as a positive control at concentrations of 0.1; 0.5; 1; 2; 5 μg/ml. The Sulforhodamine B (SRB) cytotoxicity assay showed that RHPS4 sensitivity was unchanged in U251MG and BLM −/− (IC50 was 0.56 μM in both cell lines). f Short-term cell proliferation in untreated cells, U251MG and BLM −/− , and in RHPS4-treated cells (0.5 μM) as evaluated 48 and 96 h after the first treatment. g Long-term cell proliferation in U251MG and BLM −/− untreated and RHPS4-treated cells (0.5 μM). Scale bars represent 5 μm. * p < 0.05, ** p < 0.01 (two-way ANOVA; n = 3). Error bars denote the standard deviation of the mean.

Article Snippet: Membranes were then incubated at 4 °C ON with the following primary antibodies: rabbit polyclonal anti-BLM (#A300-110A, Bethyl) (1:500); rabbit polyclonal anti-FANCJ (#B1310, Sigma-Aldrich) (1:1000), and mouse monoclonal anti-Vinculin (#V9131, Sigma-Aldrich) (1:1000).

Techniques: Staining, Immunofluorescence, Positive Control, Cytotoxicity Assay, Standard Deviation

Journal: Communications Biology

Article Title: BLM and FANCJ role in the response to G-quadruplex-dependent telomeric replicative stress

doi: 10.1038/s42003-025-09367-z

Figure Lengend Snippet: a Representative images of U251MG and siFANCJ cells immunostained using anti-BLM and anti-FANCJ antibodies (red and green signals, respectively). b The graph shows the frequency of BLM (red circles), FANCJ (green circles), and colocalization (orange circles) foci in untreated and RHPS4-treated U251MG, siSCR, and siFANCJ cells. * p < 0.05, **** p < 0.0001 (ordinary one-way ANOVA; n = 3). c Western blot showing BLM protein levels in U251MG and siFANCJ cells. d Western blot representative image and densitometric analysis of FANCJ protein level in U251MG and BLM −/− cells. * p < 0.05 (Unpaired t -test) ( n = 3). e Representative PLA images of BLM −/− , U251MG, and RHPS4-treated U251MG cells. BLM −/− cells were used as controls. f Quantification of the PLA signal was significantly higher in RHPS4-treated than in untreated cells, indicating that the treatment induced an increase in FANCJ–BLM interaction. * p < 0.05, **** p < 0.0001 (ordinary one-way ANOVA, Dunnett’s post-test; n = 3). Error bars denote the standard deviation of the mean.

Article Snippet: Membranes were then incubated at 4 °C ON with the following primary antibodies: rabbit polyclonal anti-BLM (#A300-110A, Bethyl) (1:500); rabbit polyclonal anti-FANCJ (#B1310, Sigma-Aldrich) (1:1000), and mouse monoclonal anti-Vinculin (#V9131, Sigma-Aldrich) (1:1000).

Techniques: Western Blot, Standard Deviation